Field of Use
The present invention relates generally to proteoglycans, and more specifically to biglycan variants and use of such variants in the treatment of disease.
Background Information
Biglycan is a 37 kd extracellular matrix protein. In muscle, biglycan exists in two glycoforms: a proteoglycan that bears two glucosaminoglycan (GAG) side chains and a non-glycanated form that lacks GAG chains. Both forms contain N-linked carbohydrates at two sites. Previous work showed that in mdx mice the recombinant non-glycanated version is effective in: 1) recruiting utrophin and other DAPC components to the muscle membrane; 2) improving muscle health; and 3) improving muscle function.
Biglycan is an extracellular matrix component of many parts of the skeleton including bone, cartilage, tendon, teeth and muscle. Biglycan is predominantly expressed as a proteoglycan, but a mature form lacking GAG side chains (‘nonglycanated’) has recently been shown to have specific functions in muscle, synapses and Wnt signaling in bone. The biglycan gene is on the X (and not Y) chromosome and is dysregulated in Turner (XO) and Kleinfelter's Syndromes (supernumery X) diseases, characterized by short and tall stature respectively. Biglycan deficient mice have shorter bones as well as lower bone mass (ostepenia/osteoporosis), another notable feature observed in Turner Syndrome. The mechanisms underlying the thinner and weaker bones produced without biglycan have been studied in detail and point to the fact that biglycan modulates multiple pathways critical to skeletal metabolism.
While biglycan is not needed for development of the musculoskeletal system, it is required for the maintenance of its integrity. In adult bone turnover is regulated by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. In the absence of biglycan, there is decreased bone formation due to defects in the maturation of osteogenic precursors that form bone. Bone Morphogenic Protein 2/4 (BMP-2/4), a well-known inducer of bone formation, is currently being used therapeutically to aid bone repair. Bone-derived cells depleted of biglycan have less BMP-2/4 binding and subsequently less osteogenic differentiation. It is logical to conclude that biglycan could be a prime candidate to enhance BMP-2/4 function in situations where it is commonly used such as in bone regeneration and repair after fracture or trauma.
Mice lacking biglycan also display pathologies typically associated with skeletal aging. Specifically, by three months of age, hallmark signs of osteoarthritis (OA) are evident in the mutant mice, including fissures, cell clustering and loss of the smooth articular cartilage surface on the joints. The OA is detected in all weight bearing joints as well as in the temporomandibular joint of the jaw. The effects of biglycan loss are exacerbated by depletion of the related small leucine-rich proteoglycan fibromodulin (Bgn−/0; Fmod−/− DKO). Molecular studies point to the abnormal sequestration of the potent growth factor TGF-beta in the combined absence of biglycan and fibromodulin causing it to be “unleashed” and subsequently overactive. The uncontrolled stimulation of TGF-beta in this context leads to hyper-proliferation, premature differentiation of cartilage derived cells, MMP induction and, ultimately, loss of the condyle tissue integrity.
Biglycan can also control the fate of skeletal stem cells by modulating the extracellular niche. This function was demonstrated in ECM-rich tendon tissue that harbors a cell population with stem cell features including clonogenicity, multipotency and regenerative capabilities. The combined removal of biglycan and fibromodulin caused tendon stem/progenitor cells to be hypersensitive to BMP-2. Instead of differentiating into tendon, these progenitors form multiple ectopic bones within the tendons that affect the gait of the mice. Biglycan also controls other factors critical to bone in addition to TGF-beta and BMP-2/4. In humans, a mutation in the extracellular domain of the key Wnt signaling molecule LRP-6 (R611C) causes elevated cholesterol and osteopenia. Notably, exogenous application of non-glycanated biglycan repaired the defective Wnt signaling in cells expressing mutant LRP-6. Thus, biglycan could potentially ameliorate pathologies caused by defective Wnt signaling. Taken together these findings underscore the importance of biglycan in modulating several key growth factor-mediated signaling pathways that regulate skeletal tissue architecture and function.
Biglycan also plays a role in organizing membrane architecture and function in muscle and at synapses. Muscle membranes are highly specialized to transmit force, protect the cell from contraction-induced damage and orchestrate signaling pathways required for normal function. The dystrophin- and utrophin-membrane glycoprotein complexes (DGC and UGC, respectively) link the cytoskeleton to the extracellular matrix and serve as a scaffold for signaling molecules in adult (DGC) and immature (UGC) muscle. Biglycan binds to three shared components of these complexes: the extracellular peripheral membrane protein alpha-dystroglycan and the transmembrane proteins alpha- and beta-sarcoglycan. Genetic studies show that biglycan regulates the expression of utrophin, the two sarcoglycans and an intracellular membrane-associated signaling complex comprised of dystrobrevin, syntrophins and nNOS (neuronal nitric oxide synthase) in immature muscle. Notably, dosing mice with recombinant non-glycanated biglycan (rNG-BGN) can restore the expression of several of these components to the membrane.
The role of biglycan in binding and regulating several components of DGC and UGC, coupled with the ability to deliver rNG-BGN systemically, suggested that biglycan could be a therapeutic for Duchenne Muscular Dystrophy (DMD). DMD is the most common form of muscular dystrophy and results from mutations in dystrophin—a large intracellular protein that links the actin cytoskeleton to the membrane and anchors the DGC. Notably, utrophin upregulation can compensate for dystrophin loss in mouse models of DMD (mdx; Davies). Systemically-delivered rNG-BGN recruits utrophin to the membrane and improves muscle health and function in mdx mice. The efficacy of the non-glycanated form (i.e. lacking GAG side chains) in this therapeutic approach is most likely based on two reasons. First, this form can be readily manufactured in a homogeneous form. Second, the glyconated form of biglycan, proteoglycan (PG), is proinflammatory, but the non-glycanated (core) is not. A non-glycanated form of biglycan is currently in preclinical development for DMD.
Biglycan is also important for synapse stabilization. In biglycan-deficient mice, neuromuscular junctions form normally but then they become unstable about three weeks after birth. The mechanism of biglycan action at the synapses is likely to involve MuSK, a receptor tyrosine kinase that is the master regulator of synapse differentiation and maintenance. Biglycan binds to MuSK and regulates its expression in vivo. Notably, synaptic loss is one of the earliest abnormalities observed in almost all neurodegenerative diseases, including ALS (amyotrophic lateral sclerosis) and SMA (spinal muscular atrophy). Treatments that promote neuromuscular junction stability could prolong function and potentially survival in these devastating motor neuron diseases.